Novel Green Fluorescent Probe Stem From Carbon Quantum Dots for Specific Recognition of Tyrosinase in Serum and Living Cells

Tyrosinase (TYR), an important biomarker for melanoma, offered significant information early detection of melanoma and may decrease the likelihood of mortality. Therefore, this article constructed a highly sensitive and selective green fluorescent functionalized carbon quantum dots (TYR-CQDs) for tyrosinase (TYR) activity detection by one-step hydrothermal protocol utilizing catechol, citric acid and urea as precursors. The prepared TYR-CQDs illustrated excellent linear relationship and broad linear range with a low detection limit, which exhibited high accuracy and recovery in quantitative determination of TYR in human serum samples. Furthermore, the TYR-CQDs had successfully realized intracellular TYR detection owing to excellent biocompatibility, high anti-interference ability and good cellular imaging capability, suggesting the potential biomedical applications in early diagnosis of melanoma and other tyrosinase-related diseases.


Introduction
Melanoma, as a kind of malignant cutaneous cancer with a highly metastatic nature, predominantly generated from mutated melanocytes in skin, hair, and eyes [1,2]. The cutaneous melanoma has experienced a rapidly increased morbidity and fatality rate in decades, making it one of the fastest rising cancers worldwide and the leading to death from skin diseases [3][4][5]. Tyrosinase (EC 1.14.18.1), as a binuclear copper-containing phenolic oxidase enzyme, can catalyze hydroxylation of monophenols to o-diphenols or catechol to the corresponding o-quinone [6,7]. Tyrosinase plays a vital role in melanin synthesis and constitutive expression in malignant melanocytes [8,9], which have been detected overexpression and imbalance distribution in the serum of melanoma patients [10], tyrosinase is an important and significant biomarker for melanoma in clinical diagnosis [11,12]. Therefore, constructing novel methodology for the sensitive detection of tyrosinase levels in biosystems is of great important and imperative for early clinical diagnosis of melanoma and TYR-related diseases.
Owing to the biological and physiological characteristics of tyrosinase, various analytical methods had been explored for quantitative identification and detection tyrosinase activity, including spectrophotometry [13], immunoassay [14], electrochemical detection [15], colorimetry [16], high performance liquid chromatography [17], artificial ion nanochannels [18], etc. Nevertheless, most of these methods have certain limitations, including weak selectivity, poor sensitivity, long time consumption, high expense, complex instrument, and operation. Fluorescent analytical technology had drawn much attention and has been utilized as an effective medium for quantitative detection of various biomarkers in the past decades, such as ions, proteins enzymes, reactive oxygen and nitrogen species. Fluorescent probes have better application prospects compared to all these traditional analytical tools because of high selectivity, great sensitivity, rapid implementation, easy accessibility and superior bioimaging capability [19][20][21][22].
Carbon quantum dots (CQDs), as a novel type of zerodimensional carbon-based fluorescent nanomaterials, exhibited comprehensive application value in cell imaging, biosensing, drug delivery, food safety, biochemical analysis, photoelectric device, photocatalysis and other fields [23][24][25][26][27][28][29]. CQDs had attracted widespread attention due to their excellent optical properties, such as high biocompatibility, good water solubility, outstanding photostability, simple preparation, easy functionalization, and great fluorescence performance [30][31][32]. It was used in biological analysis to detect various biological molecules, including alkaline phosphatase, acetylcholinesterase, trypsin, etc. [33][34][35]. However, there were relatively few reports on the application of CQDs for TYR activity detection, especially in biosystems. In the reported studies on CQDs, some methods used strong acid (including H 2 SO 4 , HNO 3 ) for treatment, which was not environmentally friendly and had great dangers in operation. For example, Chai synthesized a dopamine-functionalized carbon quantum dots (Dopa-CQDs) in two steps using activated carbon, concentrated H 2 SO 4 and HNO 3 as raw materials for monitoring the level of TYR in melanoma cells [36]. In addition, there were CQDs synthesized for TYR detection through multi-step reactions, which were cumbersome and had extremely low yields. Wang prepared a dual-emission ratiometric fluorescent probe (APBA-QDs) for detecting TYR activity by two-step reaction with 6-hydroxycoumarin as substrate [37]. The hydrothermal method was the best preparation method because of its simple operation and environmental friendliness, and the CQDs prepared by this method has the advantages of uniform particles, good water solubility and good dispersion [38,39]. However, the CQDs currently prepared by hydrothermal method for detecting TYR activity still have the disadvantage of low fluorescence quantum yield. For example, Hu synthesized a CQDs (CQDs-Dopa) for detecting TYR activity by hydrothermal method, but its quantum The yield was only 2.1% [40]. Therefore, the preparation of CQDs with excellent performance using safe, low-cost starting materials and a simple method has great potential for TYR analysis.
In this context, we had constructed a novel tyrosinase fluorescent sensor (TYR-CQDs) by one-step hydrothermal procedure with catechol, citric acid and urea. TYR-CQDs illustrated good water solubility, high sensitivity, great stability, strong anti-interference ability and excellent biocompatibility. Based on the excellent linear relationship and low detection limit, TYR-CQDs was successfully utilized for the quantitative analysis of TYR in human serum samples and realtime monitoring intracellular TYR by confocal fluorescence microscopy. This research offered a facile synthesized procedure and novel methodology for tyrosinase activity detection and CQDs fabrication, which illustrated potential importance for both fundamental researches in biological systems and practical applications in the clinical diagnosis of melanoma.

Instrumentation and Characterization
Transmission electron microscopic (TEM) and high-resolution TEM (HRTEM) images were measured by Tecnai G2F20 microscope. The size distribution and zeta potential of TYR-CQDs were determined by a Zetasizer Nano ZS instrument (Malvern, UK). Fourier transform infrared (FT-IR) spectra were collected by a Nicolet iS10 FT-IR spectrometer. The UV-vis spectra were recorded on UV-3600 spectrophotometer. The fluorescence spectra were acquired by the Hitachi F-4600 fluorescence spectrometer. X-ray diffraction (XRD) analysis data was collected by a TD-3500 X-ray diffractometer. X-ray photoelectron spectroscopy (XPS) survey data was obtained by a Thermo Escalab 250xi. The cellular imaging was carried out using Nikon Ti2-E confocal laser fluorescence microscopy.

Synthesis of TYR-CQDs
The green-emitting TYR-CQDs were prepared by the classical one-pot hydrothermal method. Firstly, 0.24 g citric acid (1.25 mmol)、0.15 g urea (2.50 mmol) and 0.6 g catechol (5.45 mmol) were dissolved in 10 mL anhydrous ethanol, and then the mixture was poured into a Tefon-lined stainless-steel autoclave and heated at 185 ℃ for 10 h. After the autoclave was naturally cooled down to room temperature, the reaction mixture was centrifuged at 8500 rpm for 20 min to remove solids, and ethanol solvent was segregated by a rotary evaporator. Subsequently, the product was ultrasonically dispersed in 20 mL deionized water. To further purify TYR-CQDs, the solution was dialyzed through a dialysis membrane (MWCO = 500 Da) for 24 h after filtering by a 0.22 μm microporous membrane. Finally, the purified TYR-CQDs powder was obtained after vacuum freeze-drying for 24 h, which was collected and stored at 4 °C for further characterization and application.

Fluorescence Assay of TYR with TYR-CQDs
All the fluorescence detection was performed in phosphate buffered.

Cellular Imaging
The Hela cells were cultured in confocal microscope dish with 1.0 mL fresh culture medium for 24 h. After removing the culture medium, 1.5 mL complete medium with 70 μg/ mL TYR-CQDs was added into confocal dish for 4 h. Subsequently, different concentration of TYR was added for 1 h after the excess medium was removed and each dish was washed three times with PBS. Subcellular image analysis was monitored and collected at 500-600 nm by confocal microscope at an excitation wavelength of 465 nm.

Characterization of TYR-CQDs
The morphological features and microstructures of TYR-CQDs were evaluated by transmission electron microscopy (TEM). As shown in Fig. 1A, the nanoparticles displayed uniform spherical morphology and homogeneous dispersion, which illustrated smooth surface microstructures with an average diameter of 2.8 nm. The High-resolution TEM (HRTEM) image (inset of Fig. 1A) revealed the interlayer lattice spacing was 0.34 nm, which represented the (002) diffraction planes of graphitic structure [41].
The dynamic light scattering (DLS) was utilized to analyze the size distributions and dispersion of TYR-CQDs, which revealed normal distribution ranging from 1.74 to 7.53 nm with an average hydrodynamic size of 3.72 nm (Fig. 1B). The broader size distributions attributed to nanoparticles swelling process in aqueous solution and small amount aggregation by abundant hydrophilic amino-group, carboxyl, and hydroxyl groups on the surface of TYR-CQDs.
The zeta potential of TYR-CQDs was measured by the Malvern potentiometer and displayed -57.05 mV (Fig. S1), which was attributed to the presence of a large number of surface phenolic hydroxyl, carboxyl and amino groups. The above analysis data demonstrated that TYR-CQDs indicated excellent water solubility and dispersion.
The FT-IR spectroscopy was measured to characterize the surface structures and functional groups of the nanosensor (Fig. 1C). The characteristic peaks at 3430 cm −1 represented the stretching vibration of oxygen-hydrogen (O-H). The sharp peaks at 1256 cm −1 and 1190 cm −1 represented carbon-oxygen (C-O) stretching vibration. The absorption band at a wavelength of 1600 cm-1 was caused by the aromatic carbon-oxygen double bonds (C = C), indicating that the phenolic hydroxyl groups in catechol were completely retained on the surface of TYR-CQDs. The characteristic peaks at 3190 cm −1 and 1356 cm −1 represented the stretching vibration of nitrogen-hydrogen (N-H) and carbon-nitrogen (C-N) bonds. The absorption peak at 2976, 2750 and 1450 cm −1 correspond to stretching vibration of carbon-hydrogen bond (C-H). The characteristic stretching vibration of carbon-oxygen double bond (C = O) and carbon-carbon bond (C-C) were observed at 1696 cm −1 and 1040 cm −1 , respectively. The characteristic absorption peak near 2350 cm −1 was caused by CO 2 . The infrared spectra further demonstrated phenolic hydroxyl, carboxyl and amino groups retained on the surface framework of TYR-CQDs.
Furthermore, X-ray diffraction (XRD) was utilized to explore crystallinity of TYR-CQDs (Fig. 1D). The broad peak at 2θ = 21.67 • was ascribed to the (002) plane of graphite carbon [42], which coincided with HRTEM analysis and demonstrated TYR-CQDs possessed a graphite-like structure.

Optical Properties of TYR-CQDs
The as-prepared TYR-CQDs exhibited distinctive optical properties in UV-Vis and fluorescence spectra. The UV-Vis spectra of TYR-CQDs displayed double absorption peaks at 275 and 342 nm (Fig. 3A), which attributed to π − π* transition of the carbon carbon double bonds (C = C) from benzene ring skeleton and n-π* transition of the carbon-oxygen (C = O) double bonds from carboxyl group, respectively [50,51]. The UV-Vis optical features resulted from a considerable long aromatic conjugated system and aromatic hydrocarbons on the surface of TYR-CQDs. Meanwhile, the fluorescence emission spectrum exhibited a strong and relatively constant peak at 550 nm with the excitation wavelength ranging from 435 to 495 nm, which demonstrated a significant excitation-independent characteristic and only one specific  (Fig. 3B). The fluorescence emission illustrated the strongest intensity while excited at 465 nm, which was determined as the excitation wavelength of TYR-CQDs. The fluorescence quantum yield of TYR-CQDs was determined to be 12.8% by the comparative method with quinine sulfate in 0.1 M H 2 SO 4 solution, which illustrated great potential in high sensitivity and quick response toward TYR. The calculation process and method of quantum yield was exhibited in the experimental procedure of supporting information.
Moreover, the photostability and chemical stability of TYR-CQDs were particularly significant factors that should be considered in constructing fluorescent sensors. Firstly, photobleaching experiment was performed to investigate the photostability of TYR-CQDs (Fig. S2). The experimental results exhibited no obvious fluorescence fluctuation after 60 min irradiation with xenon lamp, which demonstrated that TYR-CQDs possessed excellent photostability and strong photobleaching resistance.
Additionally, the fluorescence interferences from ionic strength were also investigated by different concentrations of sodium chloride (NaCl). As shown in Fig. S3, the photoluminescence didn't illustrate any significant fluctuation even at high concentration of NaCl (1.0 mol/L), which further demonstrated the high photostability and anti-interface ability to ionic strength.
The interference from pH was further investigated to evaluate the chemical stability and photostability. However, the fluorescence intensity fluctuated as the pH increased from 4 to 10, while it was relatively stable in the physiological pH range from 6 to 9 (Fig. S4), which implied that the fluorescence intensity didn't receive any influences in biological systems. The fluorescence intensity of TYR-CQDs decreased under acidic or alkaline conditions, which was due to the reversible protonation reaction of the amino and carboxyl groups rich in its surface [52]. Under acidic conditions, -NH 2 was protonated into -NH 3 + , while under alkaline conditions, -COOH was converted into -COO − through deprotonation. The change of TYR-CQDs surface groups led to the attenuation of fluorescence intensity. All these experimental results proved that TYR-CQDs maintained excellent photostability and potential application in biological systems and complex environment. To further evaluate the selectivity of TYR-CQDs towards TYR activity, the interference factors of various proteins (SPI, PPI, OVA, BSA) and enzymes (GOX, LYS, ALP), various amino acids (His, Ser, Ala, Thr, Tyr, Cys, Cys-Cys) were examined and evaluated (Figs. 3C, D). The fluorescence intensity was almost kept constant (< 5% decrease) except for TYR (> 87% decrease), exhibiting little chemical response of TYR-CQDs towards the above interference factors. The interfering experiments demonstrated that TYR-CQDs possessed high selectivity toward TYR and strong anti-interference capability to other biologically active substances. Therefore, the experiments implied that most biological substances can coexist in TYR-CQDs solution for TYR activity detection without any interference, which indicated great potential for widespread application in biological systems and complex environment.

Fluorescence Assay of TYR based on TYR-CQDs
The time-dependent experiment of TYR-CQDs was launched and recorded to evaluate fluorescence quenching rate by TYR. The experimental results illustrated the fluorescence intensity of TYR-CQDs quenched more than 87% in 50 min and no apparent change in emission wavelength with the increased incubation time of TYR (Fig. 4A), implying the oxidation reaction between TYR-CQDs and tyrosinase reached an equilibrium state in 50 min under a high sensitivity. Meanwhile, the bright green cuvette could be observed under ultraviolet lamp, while it attenuated to almost colorless after incubation with TYR (inset of Fig. 4A). The obvious color change indicated the potential of TYR-CQDs for detecting TYR activity through the naked-eye.
In addition, the fluorescence performance of TYR-CQDs toward different activities of TYR was evaluated. As  Fig. 4B, the fluorescence intensity of TYR-CQDs continued to decrease with the increased addition of TYR, which was almost completely quenched while the TYR concentration was as high as 7200 U/L. The result attributed to the surface bisphenolic hydroxyl of TYR-CQDs was oxidized and catalyzed into o-quinone derivatives by TYR (Scheme 1), which induced photoinduced electron transfer of TYR-CQDs and fluorescence quenching.
The sensing linear relationship between TYR-CQDs and different concentrations of TYR was investigated and fitted. The fluorescence titrations with different concentrations of TYR (from 0 to 1200U/L) exhibited a gradual quenching procedure (Fig. 4C).
The quenching efficiency (F 0 -F) of TYR-CQDs was highly linear correlated with TYR activity, which could be fitted into linear equation as follows: F 0 -F = 1.229 [TYR] + 39.45, R 2 = 0.9945, where F 0 and F were the fluorescence intensity without TYR and with TYR, respectively. (F 0 -F) represented the fluorescence change while [TYR] represented tyrosinase activity or concentrations. According to 3σ (signal-to-noise ratio) standard calculation illustrated that the limit of detection (LOD) was 33.21 U/L, manifesting a strong potential and prospect to track trace TYR in biosystem by TYR-CQDs. The excellent linear relationship demonstrated that TYR-CQDs could be utilized as a novel analysis tool to sensitively detect TYR activity, which possessed a promising applicability in the field of trace tyrosinase detection.

TYR Analysis in Human Serum Sample
To evaluate the accuracy and repeatability of quantitative detection in the complicated biological environment, the TYR-CQDs was utilized to detect TYR activity in fresh human serum. The serum samples with different standard concentrations of TYR (50,200, 600 and 1000 U/L) were added in TYR-CQDs system, then the fluorescence intensity change was recorded and the analytical recovery rate was conducted. The fluorescence spectra of TYR with different concentrations in serum were shown in Fig. S5. It can be found that the fluorescence intensity decreased with the increase of TYR concentration, and then the recovery and accuracy were analyzed through calculation. As illustrated by the summarized data in Table 1, the average recoveries of TYR ranged from 95.28% to 103.70% and the relative standard deviations (RSD) was lower than 1.5%, demonstrating an excellent accuracy and repeatability of TYR-CQDs for trace tyrosinase detection in actual human serum samples. Therefore, the novel fluorescent nanosensor illustrated great potential for TYR activity detection in actual complex biological samples and provided credible evidence for early clinical diagnosis of tyrosinase-related diseases.
The specific analytical features of TYR-CQDs were evaluated by comparison with other reported fluorescent

Table 2
Comparison with different fluorescent sensors of TYR sensors. The analytical methods and previously reported tyrosinase sensors were summarized in Table 2. Compared to complicated synthetic methods and analysis equipment, the TYR-CQDs illustrated obvious advantages, including one-step hydrothermal synthesis, good water solubility, excellent biocompatibility, low detection limit, high selectivity and repeatability, exhibiting great superiority and potential for TYR activity detection in actual complex biological samples.

Biocompatibility and Biosensing Experiment
To explore the possible application of TYR-CQDs in tyrosinase detection in vitro, Hela cell line was chosen for further cytotoxicity evaluation and intracellular TYR detection (Fig. 4D). The MTT results indicated that Hela cells remained at a comparatively high viability (> 93%) even while the concentration of TYR-CQDs was up to 100 μg/ mL, indicating that the TYR-CQDs contained excellent biocompatibility and great potential application for bioimaging in living cells. The confocal fluorescence imaging experiment for detecting intracellular TYR was further performed. As shown in Fig. 5, the Hela cells displayed strong green fluorescence after incubation with TYR-CQDs (70 μg/ mL) for 4 h, which illustrated clear outline and complete morphology under bright green fluorescence. The confocal images demonstrated that TYR-CQDs possessed excellent transmembrane permeability and cellular uptake velocity. The fluorescence intensity of TYR-CQDs in Hela cells quenched gradually with the increased addition of TYR, which demonstrated that the fluorescence change process in vivo was consistent with the extracellular sample analysis. The confocal results indicated that TYR-CQDs had good biocompatibility and successfully achieved trace TYR detection in living cells. Therefore, TYR-CQDs as a nanosensor could be potentially utilized to real-time monitor TYR activity in vivo and provided evidence for the prevention and early diagnosis of TYRassociated diseases.

The Possible Mechanism for TYR-CQDs Response to TYR
In order to explore the quenching mechanism, the fluorescence lifetime before and after adding TYR was used to distinguish the quenching type of the process. As shown in Fig. S6, the fluorescence lifetime of TYR-CQDs was 3.32 ns, and the fluorescence lifetime had not changed after adding TYR, which indicated that there was a static quenching between TYR-CQDs and TYR [61].
The expected mechanism and catalytic oxidation process of the TYR-CQDs response toward TYR were illustrated in Scheme 1. The fluorescence analysis of TYR activity performed with organic molecules sensors have attracted widespread attention and reports [37, 62,63], which considered the catalytic oxidation procedure and photoinduced electron-transfer (PET) process as fluorescence recognized mechanisms [40,[64][65][66].
Based on the relevant literature and previous research on TYR activity detection [40,53,67,68], the TYR-CQDs response to TYR was caused by the specific catalytic oxidation reaction between surface catechol groups and TYR, which was estimated to generate o-quinone structure and induce photoinduced electron-transfer (PET), and then resulting in fluorescence quenching (Scheme 1).
In order to further verify the mechanism, carbon quantum dots (CQDs) were synthesized by p-aminophenol (PAP-CDs) and PAP-CDs + TYR (PAP-CDs incubated with TYR) were utilized as control research. It can be seen from Fig. S7 that after adding TYR (7200 U/L), the fluorescence intensity of PAP-CDs only decreased by 8%, while the quenching efficiency of TYR-CQDs reached 83% (Fig. 4B). In addition, the infrared spectrum of PAP-CDs and PAP-CDs + TYR in the control group were basically similar without any obvious changes, as illustrated in Fig. S8. The above analysis showed that the selectivity of TYR to the catalytic oxidation of catechol group was much higher than that of mono phenol hydroxyl group.
Compared with the control group, the infrared spectrum of TYR-CQDs and TYR-CQDs + TYR (TYR-CQDs incubated with TYR) illustrated significant and notable change. After incubation with TYR, the oxygen-hydrogen (O-H) stretching vibration peak of TYR-CQDs at 3420 cm −1 disappeared. The above IR analysis results demonstrated that the catechol groups on the surface of TYR-CQDs underwent catalytic oxidation reaction with TYR to form o-quinone structure (Scheme 1).

Conclusion
In summary, a novel functional TYR-CQDs were firstly constructed and synthesized via a one-step hydrothermal strategy utilizing catechol, citric acid and urea as precursors. With excellent fluorescence properties, outstanding water solubility, superior biocompatibility and exceptional photostability, the TYR-CQDs was exploited to be a fluorescence sensor for intracellular tyrosinase detection. The prepared TYR-CQDs exhibited high selectivity for tyrosinase over other biological substances, including various intracellular protease, amino acids and even in real human serum sample analysis, which attributed to the catalytic oxidation procedure and photoinduced electron-transfer (PET) process mechanism. The quantitative analysis methodology of TYR activity was established and illustrated an excellent linear relationship and low detection limit. In addition, the prepared TYR-CQDs had successfully achieved the TYR detection in human serum samples and fluorescence imaging of living cells with high accuracy and recovery, which demonstrated this biosensor presented great prospect in multiple applications and a huge potential in tyrosinase-related disease monitoring and early clinical diagnosis.